Oxidative stress in patients with phenylketonuria.

Phenylketonuria (PKU) is an autossomal recessive disease caused by phenylalanine-4-hydroxylase deficiency, which is a liver-specific enzyme that catalyzes the hydroxylation of l-phenylalanine (Phe) to l-tyrosine (Tyr). The deficiency of this enzyme leads to the accumulation of Phe in the tissues and plasma of patients. The clinical characterization of this disease is mental retardation and other neurological features. The mechanisms of brain damage are poorly understood. Oxidative stress is observed in some inborn errors of intermediary metabolism owing to the accumulation of toxic metabolites leading to excessive free radical production and may be a result of restricted diets on the antioxidant status. In the present study we evaluated various oxidative stress parameters, namely thiobarbituric acid-reactive species (TBA-RS) and total antioxidant reactivity (TAR) in the plasma of PKU patients. The activities of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were also measured in erythrocytes from these patients. It was observed that phenylketonuric patients present a significant increase of plasma TBA-RS measurement, indicating a stimulation of lipoperoxidation, as well as a decrease of plasma TAR, reflecting a deficient capacity to rapidly handle an increase of reactive species. The results also showed a decrease of erythrocyte GSH-Px activity. Therefore, it is presumed that oxidative stress is involved in the pathophysiology of the tissue damage found in PKU.

Reduction of large neutral amino acid levels in plasma and brain of hyperleucinemic rats.

Neurological dysfunction is common in patients with maple syrup urine disease (MSUD). However, the mechanisms underlying the neuropathology of this disorder are poorly known. In the present study we investigated the effect of acute hyperleucinemia on plasma and brain concentrations of amino acids. Fifteen-day-old rats were injected subcutaneously with 6 micromol L-leucine per gram body weight. Controls received saline in the same volumes. The animals were sacrificed 30--120 min after injection, blood was collected and their brain rapidly removed and homogenized. The amino acid concentrations were determined by HPLC using orthophtaldialdehyde for derivatization and fluorescence for detection. The results showed significant reductions of the large neutral amino acids (LNAA) L-phenylalanine, L-tyrosine, L-isoleucine, L-valine and L-methionine, as well as L-alanine, L-serine and L-histidine in plasma and of L-phenylalanine, L-isoleucine, L-valine and L-methionine in brain, as compared to controls. In vitro experiments using brain slices to study the influence of leucine on amino acid transport and protein synthesis were also carried out. L-Leucine strongly inhibited [14C]-L-phenylalanine transport into brain, as well as the incorporation of the [14C]-amino acid mixture, [14C]-L-phenylalanine and [14C]-L-lysine into the brain proteins. Although additional studies are necessary to evaluate the importance of these effects for MSUD, considering previous findings of reduced levels of LNAA in plasma and CSF of MSUD patients during crises, it may be speculated that a decrease of essential amino acids in brain may lead to reduction of protein and neurotransmiter synthesis in this disorder.

Inhibition of the mitochondrial respiratory chain complex activities in rat cerebral cortex by methylmalonic acid.

Propionic and methylmalonic acidemic patients have severe neurologic symptoms whose etiopathogeny is still obscure. Since increase of lactic acid is detected in the urine of these patients, especially during metabolic decompensation when high concentrations of methylmalonate (MMA) and propionate (PA) are produced, it is possible that cellular respiration may be impaired in these individuals. Therefore, we investigated the effects of MMA and PA (1, 2.5 and 5mM), the principal metabolites which accumulate in these conditions, on the mitochondrial respiratory chain complex activities succinate: 2,6-dichloroindophenol (DCIP) oxireductase (complex II); succinate: cytochrome c oxireductase (complexII+CoQ+III); NADH: cytochrome c oxireductase (complex I+CoQ+complex III); and cytochrome c oxidase (COX) (complex IV) from cerebral cortex homogenates of young rats. The effect of MMA on ubiquinol: cytochrome c oxireductase (complex III) and NADH: ubiquinone oxireductase (complex I) activities was also tested. Control groups did not contain MMA and PA in the incubation medium. MMA significantly inhibited complex I+III (32-46%), complex I (61-72%), and complex II+III (15-26%), without affecting significantly the activities of complexes II, III and IV. However, by using 1mM succinate in the assay instead of the usual 16mM concentration, MMA was able to significantly inhibit complex II activity in the brain homogenates. In contrast, PA did not affect any of these mitochondrial enzyme activities. The effect of MMA and PA on succinate: phenazine oxireductase (soluble succinate dehydrogenase (SDH)) was also measured in mitochondrial preparations. The results showed significant inhibition of the soluble SDH activity by MMA (11-27%) in purified mitochondrial fractions. Thus, if the in vitro inhibition of the oxidative phosphorylation system is also expressed under in vivo conditions, a deficit of brain energy production might explain some of the neurological abnormalities found in patients with methylmalonic acidemia (MMAemia) and be responsible for the lactic acidemia/aciduria identified in some of them.

Quinolinic acid inhibits glutamate uptake into synaptic vesicles from rat brain.

Quinolinic acid (QA) is an endogenous and potent neurotoxin associated with the neurotoxicity of various common diseases. The uptake of neurotransmitters into synaptic vesicles is an important event involved in the storage and release of neurotransmitters by vesicles. The influence of QA on the uptake of glutamate, GABA and glycine into rat brain synaptic vesicles was investigated. QA (0.3-10 mM) significantly inhibited (>50%) the uptake of glutamate into synaptic vesicles, whereas QA at concentrations up to 10 mM had no significant effect on GABA or glycine uptake. Such results indicate that QA is able to selectively inhibit the vesicular uptake of glutamate, without interfering with the uptake of the inhibitory neurotransmitters GABA and glycine. These findings might be related to the neurotoxic effects of QA in the brain.

Propionic and L-methylmalonic acids induce oxidative stress in brain of young rats.

The in vitro effects of propionic and L-methylmalonic acids on some parameters of oxidative stress were investigated in the cerebral cortex of 21-day-old rats. Chemiluminescence, thiobarbituric acid-reactive substances (TBA-RS) and total radical-trapping antioxidant capacity (TRAP) were measured in brain tissue homogenates in the presence of propionic or L-methylmalonic acids at concentrations ranging from 1 to 10mM. Both acids significantly increased chemiluminescence and TBA-RS and decreased TRAP, indicating a simulation of lipid peroxidation and a reduction of tissue antioxidant potential. Other organic acids tested which accumulate in some organic acidemias (suberic, sebacic, adipic, 3-methylglutaric and 4-hydroxybutyric acids) did not affect these parameters. This study provides evidence that free radical generation may participate in the neurological dysfunction of propionic and methylmalonic acidemias.

Effects of methylmalonic and propionic acids on glutamate uptake by synaptosomes and synaptic vesicles and on glutamate release by synaptosomes from cerebral cortex of rats.

Neurological dysfunction is common in patients with methylmalonic and propionic acidemias. However, the mechanisms underlying the neuropathology of these disorders are far from understood. In the present study we investigated the in vitro effects of methylmalonic (MMA) and propionic (PA) acids at various concentrations (1 microM-5 mM) on three parameters of the glutamatergic system, namely the basal and potassium-induced release of L-[3H]glutamate by synaptosomes, Na+-dependent L-[3H]glutamate uptake by synaptosomes and Na+-independent L-[3H]glutamate uptake by synaptic vesicles from cerebral cortex of male adult Wistar rats. The results showed that MMA significantly increased potassium-induced but not basal L-[3H]glutamate release from synaptosomes with no alteration in synaptosomal L-[3H]glutamate uptake. A significant reduction of L-[3H]glutamate incorporation into vesicles caused by MMA was also detected. In contrast, PA had no effect on these parameters. These findings indicate that MMA alters the glutamatergic system. Although additional studies are necessary to evaluate the importance of these observations for the neuropathology of methylmalonic acidemia, it is possible that the effects elicited by MMA may lead to excessive glutamate concentrations at the synaptic cleft, a fact that may explain previous in vivo and in vitro findings associating MMA with excitotoxicity.

Effects of histidine and imidazolelactic acid on various parameters of the oxidative stress in cerebral cortex of young rats.

Histidinemia is an inherited metabolic disorder caused by deficiency of histidase activity, which leads to tissue accumulation of histidine and its derivatives. Affected patients usually present with speech delay and mental retardation, although asymptomatic patients have been reported. Considering that the pathophysiology of the neurological dysfunction of histidinemia is not yet understood and since histidine has been considered a pro-oxidant agent, in the present study we investigated the effect of histidine and one of its derivatives, l-beta-imidazolelactic acid, at concentrations ranging from 0.1 to 10 mM, on various parameters of oxidative stress in cerebral cortex of 30-day-old Wistar rats. Chemiluminescence, total radical-trapping antioxidant potential (TRAP), thiobarbituric acid reactive substances (TBA-RS), and the activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) were measured in tissue homogenates in the presence of l-histidine or l-beta-imidazolelactic acid. We observed that l-histidine provoked an increase of chemiluminescence and a reduction of TRAP at concentrations of 2.5 mM and higher, while TBA-RS measurement, GSH-Px, CAT and SOD activities were not affected. Furthermore, l-beta-imidazolelactic acid provoked antioxidant effects at high concentrations (5-10 mM) as observed by the reduction of chemiluminescence, although this compound enhanced chemiluminescence at low concentrations (0.5-1 mM). These results suggest that in vitro oxidative stress is elicited by histidine but only at supraphysiological concentrations.

Inhibition of glutamate uptake into synaptic vesicles of rat brain by the metabolites accumulating in maple syrup urine disease.

Maple syrup urine disease is an inherited metabolic disorder characterized by tissue accumulation of branched-chain amino acids and their corresponding keto acids in the affected children. Although this disorder is predominantly characterized by neurological symptoms, only few studies were carried out to investigate its neuropathology. In this study we investigated the effect of the metabolites accumulating in maple syrup urine disease on the in vitro uptake of [3H]glutamate by synaptic vesicles of rat brain. Synaptic vesicle preparations from whole brain of male adult Wistar rats (200-250 g) were incubated with the branched-chain amino acids and their corresponding keto acids at final concentrations ranging from 0.25 to 10 mM for the determination of glutamate uptake. Glutamate uptake was significantly inhibited by L-leucine, L-isoleucine, L-2-ketoisocaproic acid and L-2-keto-3-methylvaleric acid by approximately 60%, whereas L-valine and L-2-ketoisovaleric acid showed no effect. We also verified that the metabolites probably act by competitive inhibition. Therefore, it is possible that extracellular glutamate levels may be increased in maple syrup urine disease and that excitotoxicity may be involved in the neuropathology of this disorder.

Inhibition of in vitro CO2 production and lipid synthesis by 2-hydroxybutyric acid in rat brain.

2-Hydroxybutyric acid appears at high concentrations in situations related to deficient energy metabolism (e.g., birth asphyxia) and also in inherited metabolic diseases affecting the central nervous system during neonatal development, such as "cerebral" lactic acidosis, glutaric aciduria type II, dihydrolipoyl dehydrogenase (E3) deficiency, and propionic acidemia. The present study was carried out to determine the effect of 2-hydroxybutyric acid at various concentrations (1-10 mM) on CO2 production and lipid synthesis from labeled substrates in cerebral cortex of 30-day-old Wistar rats in vitro. CO2 production was significantly inhibited (30-70%) by 2-hydroxybutyric acid in cerebral cortex prisms, in total homogenates and in the mitochondrial fraction. We also demonstrated a significant inhibition of lipid synthesis (20-45%) in cerebral cortex prisms and total homogenates in the presence of 2-hydroxybutyric acid. However, no inhibition of lipid synthesis occurred in homogenates free of nuclei and mitochondria. The results indicate an impairment of mitochondrial energy metabolism caused by 2-hydroxybutyric acid, a fact that may secondarily lead to reduction of lipid synthesis. It is possible that these findings may be associated with the neuropathophysiology of the situations where 2-hydroxybutyric acid is accumulated.

Chronic postnatal administration of methylmalonic acid provokes a decrease of myelin content and ganglioside N-acetylneuraminic acid concentration in cerebrum of young rats.

Levels of methylmalonic acid (MMA) comparable to those of human methylmalonic acidemia were achieved in blood (2-2.5 mmol/l) and brain (1.35 umol/g) of rats by administering buffered MMA, pH 7.4, subcutaneously twice a day from the 5th to the 28th day of life. MMA doses ranged from 0.76 to 1.67 umol/g as a function of animal age. Control rats were treated with saline in the same volumes. The animals were sacrificed by decapitation on the 28th day of age. Blood was taken and the brain was rapidly removed. Medulla, pons, the olfactory lobes and cerebellum were discarded and the rest of the brain ("cerebrum") was isolated. Body and "cerebrum" weight were measured, as well as the cholesterol and triglyceride concentrations in blood and the content of myelin, total lipids, and the concentrations of the lipid fractions (cholesterol, glycerolipids, phospholipids and ganglioside N-acetylneuraminic acid (ganglioside-NANA)) in the "cerebrum". Chronic MMA administration had no effect on body or "cerebrum" weight, suggesting that the metabolites per se neither affect the appetite of the rats nor cause malnutrition. In contrast, MMA caused a significant reduction of plasma triglycerides, but not of plasma cholesterol levels. A significant diminution of myelin content and of ganglioside-NANA concentration was also observed in the "cerebrum". We propose that the reduction of myelin content and ganglioside-NANA caused by MMA may be related to the delayed myelination/cerebral atrophy and neurological dysfunction found in methylmalonic acidemic children.

Detection of inborn errors of metabolism in unselected patients from pediatric intensive care units in Porto Alegre, Brazil: evaluation of screening techniques.

1. The present study provides an analysis of the interpretation and usefulness of mass biochemical urine screening tests currently applied to a population of severely ill children consisting of 232 unselected individuals, under various medications, held in intensive care units. 2. Testing for glycosaminoglycans by the cetyltrimethylammonium bromide reaction is of little benefit to this population. The Ehrlich reaction, used to detect porphobilinogen, should be reserved for cases which present clinical symptoms. Owing to the large number of false-positives and because chromatography or electrophoresis can easily be used to detect tyrosine, it is suggested that the nitrosonaphthol test be abandoned. The cyanide nitroprusside reaction should be used when chromatography or electrophoresis suggests cystinuria, homocystinuria or beta-mercapto lactate disulfiduria. 3. Because of its high sensitivity in detecting reducing substances, the Clinitest is useful for selecting samples to be investigated further by sugar chromatography. 4. Given the relatively high frequency of organic acidurias, particularly methylmalonic aciduria, in intensive care unit populations, it is suggested that the p-nitroaniline reaction be incorporated into the battery of chemical urine tests. 5. A comparison between paper chromatography and high voltage paper electrophoresis in terms of effective analysis of amino acid patterns demonstrated that chromatography is preferential, since this is as sensitive as electrophoresis, does not require a special apparatus and permits the simultaneous use of plasma and urine from the same patient, thus facilitating the interpretation of amino acid patterns.

Inborn errors of metabolism. Sensitivity of screening tests in high risk patients.

We estimated the sensitivity of a screening procedure (SP) for inborn errors of metabolism (IEM) in 566 referred, high-risk patients. The 143 (25.3% of the total sample) patients with initial abnormal results in at least one screening test (ST) were recalled for further investigations. An IEM was diagnosed in 40.6 percent of the 106 patients who came for reevaluation. In 114 of the remaining 423 patients who had normal initial ST, an IEM was still suspected on basis of clinical, radiological, and/or laboratory findings and was confirmed in 30 of such patients (5.3% of the total sample and 7.1% of the patients with normal results in the SP). The sensitivity of the SP was estimated maximally as 67.4 percent and the efficiency as 80.4 percent. Twenty-five of the 30 cases undetected with the SP were patients with sphingolipidoses. The simple inclusion of thin-layer chromatography of urinary oligosaccharides in the SP should allow the detection of at least one half of these cases, increasing its sensitivity by 14.1 percent and its efficiency by 4.6 percent. In at least 7.1 percent of patients with an initial normal ST, an IEM was detected. These would have remained undiagnosed if the limitations of the SP employed had not been fully understood.

[Börjeson-Forssman-Lehmann syndrome: report of a case]. / Síndrome de Börjeson-Forssman-Lehmann: relato de um caso.

We present a case of Börjeson-Forssman-Lehmann syndrome characterized by the presence of hypogonadism, grotesque facies, microcephaly, large ears, obesity and convulsions. In addition to these findings, which were detected also in the other patients described in the literature, the present patient showed hyperglycemia and aminoaciduria. In our opinion, the presence of these alterations and the poor evolution of the patient, with death occurring during the first year of life, may contribute to the amplification of the phenotypic spectrum of the Börjeson-Forssman-Lehmann syndrome.

Investigation of oxidative stress parameters in treated phenylketonuric patients.

Phenylketonuria (PKU) is the most frequent disturbance of amino acid metabolism being caused by severe deficiency of phenylalanine hydroxylase activity. Untreated PKU patients present severe mental retardation whose pathophysiology is not completely estabilished. Despite the low-Phe diet, a considerable number of phenylketonuric patients present a mild to moderate psychomotor delay and decreased cognitive functions. In the present study we evaluated various parameters of oxidative stress namely thiobarbituric acid-reactive species (TBA-RS), total antioxidant reactivity (TAR) and activities of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) in two groups of treated PKU patients, one with well controlled and the other with high Phe blood levels in order to investigate whether blood Phe concentrations could be correlated with the extend of oxidative stress. We initially verified a marked increase of TBA-RS, and a decrease of TAR in plasma, as well as a reduction of erythrocyte GSH-Px activity which were similar in both groups of PKU patients, when compared to controls of similar ages. In contrast, CAT and SOD activities were not altered in PKU patients. These results show that oxidative stress occurs in PKU patients and that this pathogenic process is probably not directly correlated to Phe blood levels.

Na+, K+ ATPase activity is markedly reduced by cis-4-decenoic acid in synaptic plasma membranes from cerebral cortex of rats.

We have previously demonstrated that octanoic (OA) and decanoic acids (DA) inhibit Na+, K+ ATPase activity in synaptic plasma membranes from rat brain. The objective of the present study was to investigate the in vitro effects of the other metabolites that accumulate in tissues of medium-chain acyl-CoA dehydrogenase (MCAD)-deficient patients, namely cis-4-decenoic acid (cDA), octanoylcarnitine (OC), hexanoylcarnitine (HC), hexanoylglycine (HG), phenylpropionylglycine (PPG) and suberoylglycine (SG), on Na+, K+ ATPase activity in synaptic plasma membrane from cerebral cortex of 30-day-old rats. cDA, the pathognomonic compound found in this disorder, provoked the strongest inhibition on this enzyme activity at concentrations as low as 0.25 mM, whereas OC inhibited this activity at 1.0 mM and higher concentrations in a dose-dependent manner. In contrast, HC, HG, PPG and SG did not affect Na+, K+ ATPase activity. Furthermore, pre-treatment of cortical homogenates with the antioxidant enzymes catalase plus superoxide dismutase totally prevented cDA-induced Na+, K+ ATPase inhibition. We also provided evidence that cDA, as well as OA and DA, caused lipid peroxidation, which may explain, at least in part, the inhibitory properties of these compounds towards Na+, K+ ATPase. Considering that Na+, K+ ATPase is a critical enzyme for normal brain development and functioning, it is presumed that these findings, especially those regarding to the marked inhibitory effect of cDA, may be involved in the pathophysiology of the neurological dysfunction of MCAD-deficient patients.

Promotion of oxidative stress by 3-hydroxyglutaric acid in rat striatum.

The pathophysiology of the striatum degeneration characteristic of patients affected by the inherited neurometabolic disorder glutaryl-CoA dehydrogenase deficiency (GDD), also known as glutaric aciduria type I, is still in debate. We have previously reported that 3-hydroxyglutaric acid (3-OH-GA) considered the main neurotoxin in this disorder, induces oxidative stress in rat cerebral cotex. In the present work, we extended these studies by investigating the in vitro effect of 3-OH-GA, at concentrations ranging from 0.01 to 1.0 mmol/L on the brain antioxidant defences by measuring total radical-trapping antioxidant potential (TRAP), total antioxidant reactivity (TAR) and glutathione (GSH) levels, and on the production of hydrogen peroxide (H(2)O(2)), nitric oxide (NO) and malondialdehyde in striatum homogenates from young rats. We observed that TRAP, TAR and GSH levels were markedly reduced (by up to 50%) when striatum homogenates were treated with 3-OH-GA. In contrast, H(2)O(2) (up to 44%), NO (up to 95%) and malondialdehyde levels (up to 28%) were significantly increased by 3-OH-GA. These data indicate that total nonenzymatic antioxidant defences (TRAP) and the tissue capacity to handle an increase of reactive species (TAR) were reduced by 3-OH-GA in the striatum. Furthermore, the results also reflect an increase of lipid peroxidation, probably secondary to 3-OH-GA-induced free radical production. Thus, it may be presumed that oxidative stress is involved in the neuropathology in GDD.

Possible high frequency of tetrahydrobiopterin deficiency in south Brazil.

We report our experience with the deficiency of 6-pyruvoyltetrahydropterin synthase, the most common form of tetrahydrobiopterin deficiency. We investigated 5200 patients suspected of having some inborn error of metabolism in a 10-year period, and detected 30 cases (from 28 sibships) of hyperphenylalaninaemias, HPA. From these, 4 sibships (5 patients) were affected by deficiency of 6-pyruvoyltetrahydropterin synthase. All of them were ethnically mixed, with some European ancestry detected in all. The age of diagnosis ranged from 2 to 9 years, and all were initially referred for investigation by having mental retardation and seizures. All of them showed low urinary biopterin levels and a marked elevation of neopterin. Although we detected only a few cases of HPA (30), 5 cases of 6-pyruvoyltetrahydropterin account for almost 20% of this total. The literature, however, reports a proportion of around 0.5%. As the frequency of classical phenylketonuria in our region is similar to that found in Caucasians (1/12,500), we believe that the frequency of this disease in South Brazil may be higher than expected (of the order of 1/400,000). We speculate that this finding could be related to a genetic drift (or founder effect).

The role of oxidative damage in the neuropathology of organic acidurias: insights from animal studies.

Organic acidurias represent a group of inherited disorders resulting from deficient activity of specific enzymes of the catabolism of amino acids, carbohydrates or lipids, leading to tissue accumulation of one or more carboxylic (organic) acids. Patients affected by organic acidurias predominantly present neurological symptoms and structural brain abnormalities, of which the aetiopathogenesis is poorly understood. However, in recent years increasing evidence has emerged suggesting that oxidative stress is possibly involved in the pathology of some organic acidurias and other inborn errors of metabolism. This review addresses some of the recent developments obtained mainly from animal studies indicating oxidative damage as an important determinant of the neuropathophysiology of some organic acidurias. Recent data showing that various organic acids are capable of inducing free radical generation and decreasing brain antioxidant defences is presented. The discussion focuses on the relatively low antioxidant defences of the brain and the vulnerability of this tissue to reactive species. This offers new perspectives for potential therapeutic strategies for these disorders, which may include the early use of appropriate antioxidants as a novel adjuvant therapy, besides the usual treatment based on removing toxic compounds and using special diets and pharmacological agents, such as cofactors and L-carnitine.

Effects of methylmalonate and propionate on uptake of glucose and ketone bodies in vitro by brain of developing rats.

Methylmalonate (MMA) and propionate effects on glucose and ketone body uptake in vitro by brain of fed and 30-hour-fasted 15-day-old rats were studied. In some experiments cerebrum prisms were incubated in the presence of glucose and either MMA or propionate in Krebs-Ringer bicarbonate buffer, pH 7.0. In others, the incubation medium contained beta-hydroxybutyrate (HBA) or acetoacetate (AcAc) instead of glucose. We verified that MMA increased glucose uptake by brain of fasting animals, whereas propionate had no effect. In addition, MMA diminished HBA but not AcAc incorporation into brain prisms, whereas propionate provoked a diminished utilization of both ketone bodies by brain. The in vitro effect of MMA and propionate on brain and liver beta-hydroxybutyrate dehydrogenase activity was also investigated. It was shown that MMA but not propionate significantly inhibited this activity. Rats were also injected subcutaneously three times with a MMA buffered solution, and the in vivo effects of MMA on the above-mentioned parameters assessed. Results from these experiments confirmed the previously found in vitro MMA effects. Methylmalonic acidemic patients accumulate primarily methylmalonate and secondarily propionate and other metabolites in their tissues at levels comparable to those we used in our assays. Most patients who survive early stages of the disease show a variable degree of neuromotor delay. Since glucose and sometimes ketones are the vital substrates for brain metabolism, it is possible that our findings may contribute to a certain extent to an understanding of the biochemical basis of mental retardation in these patients.

Inhibition of succinate dehydrogenase and beta-hydroxybutyrate dehydrogenase activities by methylmalonate in brain and liver of developing rats.

The effects of methylmalonate (MMA) on succinate dehydrogenase (SDH) and beta-hydroxybutyrate dehydrogenase (HBDH) activities in brain and liver of 15-day-old rats were studied. The apparent Km of SDH for succinate was 0.45 mmol/L in brain and 0.34 mmol/L in liver. MMA inhibited the enzyme activity in both tissues with Ki values of 4.5 mmol/L and 2.3 mmol/L in brain and liver, respectively, and the inhibition was of the reversible competitive type. The calculated Km for HBDH with beta-hydroxybutyrate as substrate was 1.26 mmol/L in brain and 0.36 mmol/L in liver. MMA inhibited the enzyme with a Ki value of 0.015 mmol/L in brain and 0.275 mmol/L in liver. These results are probably relevant to our understanding of cerebral metabolism in methylmalonic acidaemic children, especially during ketoacidotic and hypoglycaemic crises, and may be related to the pathogenesis of cerebral dysfunction of methylmalonic acidaemia.